KR101324654B1 - Cosmetic Composition comprising the esculetin for anti-wrinkle and anti-aging effect - Google Patents

Abstract

The present invention relates to a novel use of esculletin. Esculetin of the present invention activates ERK1 / 2, p38, JNK, PI3K involved in MAPK and PI3K / Akt pathways and thus increases the expression of type I procollagen. In addition, esculletin of the present invention increases the expression of Sp1 and thus increases the expression of type I procollagen.
Therefore, the esculletin of the present invention promotes collagen synthesis and thus has excellent anti-aging activity, and in particular, the effect of improving wrinkles can be used as an effective component of the cosmetic composition.

Description

Cosmetic composition for improving wrinkles and anti-aging including esculletin as an active ingredient {Cosmetic Composition comprising the esculetin for anti-wrinkle and anti-aging effect}

The present invention relates to a novel use of esculetin. Specifically, the present invention relates to a cosmetic composition for wrinkle improvement and anti-aging characterized in that it contains esculletin.

In Korea, the aging society is rapidly coming up with the improvement of medical and public health benefits. As a result, the quality of life is being improved, and the desire for the maintenance of youth is increasing, and cosmetics, especially anti-aging The demand for cosmetics has increased.

The skin aging mechanism, which is the basis for the development of such anti-aging cosmetics, decreases the physiological function of the living body as the age increases, resulting in naturally occurring intrinsic chronological aging and exogenous progression due to prolonged ultraviolet exposure. It can be classified as photoaging (phtoaging). The clinical feature of endogenous skin aging is that the skin becomes thinner and less elastic. In addition, histological findings include thinning of the epidermis and dermis, decreased blood vessels, and decreased number of fibroblasts in the dermis. In addition, the clinical characteristics of exogenous skin aging, photoaging, is that the skin becomes rough and elastic, irregular pigmentation occurs, and deep wrinkles increase.

The root cause of this phenomenon is not yet known, but many studies have reported that it is associated with a decrease in skin collagen content (Rittie & Fisher, 2002). In other words, as for wrinkle formation, a number of results of examining basic physiological metabolic changes such as synthesis and degradation of collagen, which is a major component of skin, have been reported (Lavker RM, Cutaneous aging: chronological versus photoaging.In: Gilchrest BA (ed). Photodamage. USA: Blackwell science, Inc., 123-135 (1995).

Therefore, in order to prevent endogenous and exogenous skin aging, it is necessary to promote the synthesis of collagen in skin fibroblasts.

Generally, retinoids (RE36068), transforming growth factor (TGF-β) and betulinic acid (JP8-208424) are known as conventional materials for promoting collagen synthesis, and various natural products are known to promote collagen synthesis. .

However, retinoids are unstable compounds and have the disadvantage that irritation occurs even when a small amount is applied to the skin. In addition, most of the natural-derived raw materials have been used in the form of simple extracts, and it is difficult to continuously maintain and control the activity of the extract because it is not known exactly what effect the extracts show.

Therefore, it is necessary to find an active ingredient which is effective in improving skin wrinkles among natural products with little skin irritation.

Esculetin, on the other hand, exists extensively in plants as coumarin derivatives. It has been reported that esculletins have physiological activities such as antioxidant, anti-inflammatory and anticancer effects (Kaneko et al., 2003; Kim et al., 2008; Lee et al., 2007). In addition, esculletin inhibits lipoxygenase activity and reduces inflammation in animal models of enteritis (Fylaktakidou et al., 2004; Witaicenis et al., 2010).

However, there has been no report on the effect of promoting the collagen synthesis of esculletin, thus completing the present invention.

The present inventors completed the present invention by finding that the coumarin derivative esculletin has the above activity while studying a substance derived from natural products having anti-wrinkle or anti-aging activity.

Accordingly, an object of the present invention is to provide a cosmetic composition for anti-wrinkle and anti-aging comprising the compound of formula 1 as an active ingredient.

[Formula 1]

In order to achieve the above object, the present invention provides a cosmetic composition for anti-wrinkle and anti-aging comprising the compound of formula 1 as an active ingredient.

[Formula 1]

Since the compound of Formula 1 of the present invention promotes collagen synthesis, the skin elasticity enhancing effect and the anti-aging activity are excellent, and in particular, the wrinkle improving effect is excellent, and thus may be used as an effective component of the cosmetic composition.

Figure 1 shows the effect of esculletin type I procollagen expression in human fibroblasts.
Figure 2 shows the effect of esculletin increasing type I procollagen expression at the level of transcription.
3 shows the effect of esculletin activating MAPK and PI3K / Akt pathways in human fibroblasts.
Figure 4 shows the effect of esculletin increases the expression of type I procollagen through the MAPK and PI3K / Akt pathway.
Figure 5 shows the effect of esculletin increases the expression of the transcription factor Sp1 in human fibroblasts.

Hereinafter, the present invention will be described in detail.

Esculetin of the present invention is represented by the following formula.

[Formula 1]

The official chemical name of esculletin is 6,7-dihydroxy-2H-1-benzopyran-2-one (also known as 6,7-dihydroxycoumarin or cycorgenin).

The esculletin is generally a commercially available material or can be prepared using known synthetic methods and can be obtained by separating and purifying from plants such as wormwood.

The composition containing the esculletin as an active ingredient is excellent in anti-aging activity, in particular excellent in wrinkle improvement, prevention or treatment effect.

Specifically, the composition containing esculletin as an active ingredient promotes the synthesis of collagen by inducing the synthesis of type I procollagen (see <Examples 1 and 2>).

In the dermal layer of the skin, fibroblasts synthesize and secrete mainly type I procollagen as well as type II procollagen and other extracellular matrix proteins. Type I procollagen consists of two α1 chains and one α2 chain, which are produced by different genes, COL1A1 and COL1A2 (Shoulders & Raines, 2009). Secreted procollagens have additional peptides at the amino and carboxy terminus, which are cut by proteases in the extracellular space. The resulting tropocollagen is made from mature collagen with a triple helix structure (Shoulders & Raines, 2009).

     It has been reported that mRNA and protein levels of type I procollagen are significantly lower in older human skin than in younger human skin (Chung et al., 2001; Varani et al., 2000; Varani et al., 2006). In addition, mRNA and protein synthesis of type I procollagen per cell was decreased in skin fibroblasts of older humans than skin fibroblasts derived from young humans (Takeda et al., 1992; Varani et al., 2006). It is also reported that the number of dermal fibroblasts decreases with age (Varani et al., 2000). These studies suggest that the decrease in the number of skin fibroblasts and the expression of type I procollagen with aging is a key mechanism of skin collagen content and ultimately skin aging. The present invention was completed while studying the relationship with collagen.

In addition, the inventor further confirmed the mechanism by which the composition containing esculintine as an active ingredient promotes collagen synthesis.

Specifically, the synthesis of type I procollagen involves mitogen-activated protein kinase (MAPK) and phosphatidiylinositol 3-kinase (PI3K) / Akt pathways.

MAPKs include ERK (extracllular-regulated protein kinase) 1/2, p38, and JNK (Jun-N-terminal kinase), which are activated by phosphorylation of threonine and tyrosine residues by upper MAPK activating enzymes. Among the MAPK activating enzymes, MEK1 / 2, MKK3 / 6 and MKK4 / 7 are known to phosphorylate and activate ERK1 / 2, p38 and JNK, respectively (Pearson et al., 2001). PI3K imports Akt and Pyruvate dehydrogenase kinase (PDK) 1 into the cell membrane, and PDK1 phosphorylates and activates Akt (Leevers et al., 1999).

In one embodiment of the present invention, when treated with esculletin in human fibroblasts, it was confirmed that phosphorylation of not only ERK1 / 2, p38, JNK, but also Akt was increased, and especially observed 15 minutes after esculletin treatment. (Fig. 3).

In addition, when the MAPK activating enzyme (MEK1 / 2) and PI3K enzyme were activated, it was confirmed that the synthesis of type I procollagen in fibroblasts was increased (FIG. 4).

Thus, it can be seen that esculletin increases the expression of type I procollagen by activating the MAPK and PI3K / Akt pathways.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples. However, these examples are provided only for the purpose of illustration to help the understanding of the present invention is not limited to the scope and scope of the present invention.

Example 1 Effect of Esculetin on Synthesis of Type I Procollagen

In order to determine the effect of the type I procollagen synthesis of esculletin according to the concentration was measured by Western blotting method.

Esculetin was purchased from Tokyo Chemical Industry (Tokyo, Japan) and used.

Human dermal fibroblasts were isolated from the skin and then treated with 10% fetal bovine serum, penicillin (100 units / ml) and streptomycin (100 units / ml) using a 100 mm dish (Life technologies, Grand Island, NY, USA). Dulbecco's modified Incubated at 37 ^° C. in Eagle's medium (DMEM) medium in the presence of 5% CO ₂ .

Fibroblasts treated with 0-100 μg / ml of esculletine for 8 hours were treated with RIPA buffer (150 mM NaCl, 100 mM Tris-HCl, 1% Tween-20, 1% sodium deoxycholate and 0.1% SDS with 0.5 mM EDTA). , 1 mM PMSF, 10 mg / ml aprotinin, 10 mg / ml leupeptin and 1 mg / ml pepstatin). Proteins were extracted from fibroblast lysates, separated by SDS-PAGE and transferred to nitrocellulose membranes followed by antigen-specific primary antibodies to type I procollagen. Antibodies against type I procollagen (# sc-25974) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The antigen-antibody mass was then detected by amplification chemifluorescence (Amersham Bioscience, Boston, Mass., USA) to confirm the production of type I procollagen in fibroblasts.

β-actin was used as a control for the amount of protein.

The results obtained are shown in Figure 1A.

Referring to Figure 1A it was confirmed that the production of type I procollagen increased concentration-dependently in fibroblasts treated with esculletin.

FIG. 1B is a graph quantitatively analyzing the results obtained in FIG. 1A using densitometry. 1B, the amount of procollagen type I was increased by about 20 times compared to the control group when 10 µg / ml of esculletine was treated, and the type I procollagen compared to the control group when 100 µg / ml of esculletine was treated. It can be seen that the amount increased about 25 times.

The results indicate that esculletin increases the production of type I procollagen in a concentration-dependent manner.

Example 2 Type I Procollagen Synthesis Effect of Esculetin

Western blotting was used to determine the effect of esculletin I collagen procollagen synthesis over treatment time.

Except for treating fibroblasts with 100 μg / ml of esculletin for 0 to 24 hours, the same procedure as in Example 1 was performed.

The results obtained are shown in Figure 1C.

1C shows that type I procollagen was produced within 2 hours in fibroblasts treated with 100 μg / ml of esculletin, and the production of type I procollagen was increased in a time-dependent manner.

1D is a graph quantitatively analyzing the results obtained in FIG. 1C using densitometry. 1D shows that the amount of type I procollagen increased about 40 times compared to the control group at 2 hours after treatment with 100 μg / ml of esculletin, and the type I procollagen amount was about 80 times higher than the control group after 24 hours. It can be seen that the increase.

The results suggest that esculintin increases the production of type I procollagen in a time-dependent manner.

Example 3 Effect of Esculetin on Type I Procollagen Expression at the Transcription Level

To determine whether esculletin increases type I procollagen expression at the transcription level, COL1A1 and COL1A2 in esculletin- treated human fibroblasts The amount of transcript was measured by northern blotting.

Esculetin and human fibroblasts were used in the same manner as in Example 1.

Fibroblasts treated for 8 hours with 0-100 μg / ml esculletin were suspended in TRIzol solution (Life technologies, Grand Island, NY, USA) and dissolved by addition of chloroform. After dissolving the lysate at 12,000 xg for 15 minutes at 4 ° C, RNA was precipitated by adding the same amount of isopropanol to the supernatant. The precipitated RNA was washed once with ethanol and then dissolved in DEPC solution to extract total RNA. .

The extracted 40 mg of total RNA was electrophoresed on a 1% formaldehyde agarose gel and then transferred to a nylon membrane. Attach DNA probes for COL1A1 and COL1A2 to RNA, and then photosensitive the film to COL1A1 and COL1A2 The amount of transcript was measured by northern blotting.

DNA probes for COL1A1 and COL1A2 were developed by Dr. Dokkyo University School of Medicine in Japan. Used from Atsushi Hatamochi (Ikeda et al., 2008).

The results obtained are shown in FIG. 2AB and confirmed that esculletin increases the amount of COL1A1 transcript concentration-dependently (FIG. 2A). However, there was no significant change in the amount of COL1A2 transcript (FIG. 2B). These data suggest that esculletin increases the expression of type I procollagen primarily by increasing transcription of COL1A1 .

Example 4 Effect of Esculetin on MAPK and PI3K / Akt Pathways

Western blotting was used to determine if esculletin activates MAPK and PI3K / Akt.

Human fibroblasts were treated with 100 μg / ml of esculletin, followed by antibodies to p-ERK1 / 2 (A), p-p38 (B), p-JNK (C), and p-Akt (D) Western blotting was performed in the same manner as in Example 1 except for using.

p-ERK1 / 2 (# 9106), p-p38 (# 9211), p-JNK (# 9251), p-Akt (# 4060) antibodies were purchased from Cell Signaling Technology (Danvers, Mass., USA).

β-actin was used as a control for the amount of protein.

The obtained result is shown in FIG.

Referring to Figure 3, when treated with esculletin human fibroblasts, it was observed that the phosphorylation of Akt as well as ERK1 / 2, p38, JNK was increased, especially in about 15 minutes after esculletin treatment. These results suggest that esculletin activates the MAPK and PI3K / Akt pathways in human fibroblasts.

Example 5 Effect of MAPK and PI3K / Akt Pathway on Increasing Type I Procollagen Expression by Esculetin

Western blotting was used to determine the effects of type I procollagen synthesis through the MAPK and PI3K / Akt pathways of esculletin.

Esculetin and human fibroblasts were used in the same manner as in Example 1.

Human fibroblasts were treated with U0126 (MEK1 / 2 inhibitor) (A), SB203580 (p38 inhibitor) (B), SP600125 (JNK inhibitor) (C), LY294002 (PI3K inhibitor) (D) at 20 mM concentration. After 1 hour esculletin was added at a concentration of 100 μg / ml. After harvesting the cells hourly (0 to 24 hours), Western blot analysis was performed in the same manner as in Example 1 using type I procollagen antibody. β-actin was used as a control for the amount of protein.

The obtained result is shown in FIG.

4, esculletin significantly increased the protein level of type I procollagen in the presence of DMSO, but increased significantly in the presence of U0126, SB203580, SP600125, and LY294002. These results suggest that esculletin increases the expression of type I procollagen by activating the MAPK and PI3K / Akt pathways.

Example 6 Effect of Esculetin on Transcription Factor Sp1

Since esculletin increased the transcription of COL1A1 (Example 3), we wanted to determine what transcription factors mediate this phenomenon. To this end, human COL1A1 from the PubMed database. Sequence information on the promoter region of the gene was obtained and the promoter region within 2 kb from the transcription start site was analyzed using the AliBaba2 program. As a result, the potential binding sequences for 61 transcription factors were found on the promoter, and among them, the incidence of binding sequences for Sp1 was the highest (FIG. 5A). These results suggest that esculintin can increase the transcription of COL1A1 by activating the transcription factor Sp1.

To determine if esculletin increased the expression of Sp1, it was measured by Western blotting.

Esculetin and human fibroblasts were used in the same manner as in Example 1.

Human fibroblasts were treated with esculletin at a concentration of 100 μg / ml, and Western blotting was performed in the same manner as in Example 1 except for using an anti-Sp1 antibody.

Anti-Sp1 antibody (# sc-14027) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

β-actin was used as a control for the amount of protein.

The obtained result is shown in FIG.

5, it was observed that the protein level of Sp1 increased within 2 hours after esculletin treatment to human fibroblasts (FIG. 5B). These results suggest that esculintin can increase the transcription of COL1A1 by increasing the expression of the transcription factor Sp1.

Claims (5)

A cosmetic composition comprising the compound of formula 1 as an active ingredient and increasing the expression of type I procollagen to promote collagen synthesis.
[Chemical Formula 1]

delete The cosmetic composition according to claim 1, wherein the increase in the expression of type I procollagen increases the transcription of COL1A1. The cosmetic composition according to claim 1, wherein the increase in the expression of type I procollagen increases the expression of the transcription factor Sp1. The cosmetic composition according to claim 1, wherein the increase in the expression of type I procollagen activates the MAPK and PI3K / Akt pathways.

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